The Principle of Isoelectric Focusing The Principle of Isoelectric Focusing. A pH gradient is established in a gel before loading the sample. (A) The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric pH, the location at which they have no net charge. (B) The proteins form bands that can be excised and used for further experimentation.

The principle of IEF The IEF is a very high resolution separation method, and the pI of a protein can be measured.

Advantage of IPG strips Industrial standard (GMP) reduce variation. The chemistry of the immobiline is better controllable. The film-supported gel strips are easy to handle. The fixed gradient are consistent during IEF. Stable basic pH gradient allow reproducible results for basic proteins. High protein loads are achievable. Less protein loss during equilibration in SDS buffer.

General detection methods Coomassie Blue Dyes - commonly used - does not interfere with subsequent protein identification - inexpensive - sensitivity well below silver and fluorescent dyes Silver stain - sensitivity 10-50 times greater than CB - ability to detect 1 ng of protein - silver diammine/silver nitrate - relatively expensive (reagents/waste disposal) - high background Fluorescent Stains and Dyes - accurately determine changes in protein expression - greater sensitivity than silver stain - DIGE - cost Comassie blue routinely used to stain for proteins separated by 2d electrophoresis. Popularity continues despite the fact that they cannot accurately quantify proteins and have sensitivity well below silver and fluorescent dyes. In many instances a high degree of sensitivity is not required and unlike many silver stains, does not interfere greatly with subsequent protein identification by mass spectrometry. With a sensitivity approx 10-50 times greater than that of CB, silver stain offers the ability to detect as little as 1ng of protein. silver diammine (alkaline)/silver nitrate(acidic). There are a no of commercial kits – offering convenience and consistency. Despite the superior sensitivity, there are a no of drawbacks/limitations. Unlike CB, silver stain is relatively expensive. High background resulting from a no. of variables may cause poor resolution of protein spots. Significant protein to protein variability relative to the extent of silver deposited on the protein as a function to the degree of glycosylation. Fluorescent dyes for protein identification an attractive alternative to other detection methods. accurately determine changes in protein expression, greater sensitivity than silver stain. The staining procedure is quite simple and the resulting fluorescently labeled proteins can be imaged with a UV transluminator. Several of the new stains on the market are compatible with Mass spectrometry. Fluorescent dyes such cyanine can be used for difference gel electrophoresis (DIGE). Samples of interest are labeled with different Cy dyes and combined and subjected to 2-DE and this allows the detection of protein differences with the use of a single gel, eliminating gel-to gel variation. Cost is a drawback both for reagents and imaging equipment.

SYPRO Ruby 2-D gel stained with the SYPRO Ruby protein gel stain and the Pro-Q Emerald 300 reagent. Combined Cohn fractions II and III from cow plasma, containing primarily α - and β -globulins, were run on a 2-D gel and stained first with the Pro-Q Emerald 300 reagent (left) and then with the SYPRO Ruby protein gel stain (right). Rabilloud, T. et al. (2001) Proteomics 1, 699-704

Gel Staining Techniques Proteins were diluted into four aliquots Gel Staining Techniques Proteins were diluted into four aliquots. Gels A, B and C were stained with: Colloidal Coomassie Blue (Bio-Rad), SYPRO Ruby Red fluorescent stain (Bio-Rad) and silver, respectively. The fluorescent image was captured with a Gel Doc Station (Bio-Rad). The other two stain images were captured with an HP ScanJet 6200. Of these detection methods, the fluorescent stain and silver display more sensitivity than the Coomassie stain.

Pro-Q Diamond phosphoprotein stain can detect phosphate groups attached to tyrosine, serine or threonine residues.  It is ideal for identification of kinase targets in signal transduction pathways and for phosphoproteomic studies.  Signal intensity is linear over three orders of magnitude and correlates with the number of protein phosphates.  Stained proteins can be accurately identified by mass spectrometry. The Pro-Q Diamond phosphoprotein gel stain is particularly useful when used in conjunction with SYPRO® Ruby protein gel stain.  The SYPRO® Ruby dye quantitatively stains total proteins.  Determining the ratio of Pro-Q Diamond dye to SYPRO® Ruby dye signals provides a measure of the phosphorylation level normalized to the total amount of protein (see figure below).  Using both stains in combination, it is possible to distinguish a lightly phosphorylated, high-abundance protein from a heavily phosphorylated, low-abundance protein.

DISADVANTAGES OF 2-D PAGE...  Restricted to proteins < 106 and > 104 Da MW  Cannot detect proteins expressed at low levels  Limited to 600~800 separate spots  Gel to gel reproducibility is poor  Quantitation is poor, ± 50% or worse  Dynamic range is limited, < 10X  Analysis is not directly coupled to separation

A specific point in the 2D gel imply a specific protein with certain pI value and molecular weight. The signal intensity imply the expression of protein. Protein pattern. Protein markers.

The methods so far…..

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DIGE for the Identification of Cancer Markers Ge Zhou et al. Molecular & Cellular Proteomics 1:117–124, 2002.

A B 2D-PAGE IMAGE ANALYSIS Excise spot; elute; digest Extract peptides; MS analyze Protein identification

Dual Channel Imaging Technique (DIGE) Proteins are extracted from the cells or tissues of interest. The protein extracts are labeled with different fluorescent dyes: 3) The 2 extracts are mixed and then resolved by 2-D gel electrophoresis. Extract 1 Cy5 dye Extract 2 Cy3 dye mix

Fluorescence staining H2N- CyDye DIGE containing NHS ester active group covalently binds to the lysine residue of a protein via an amide linkage.

The DIGE technology platform

The DIGE technology platform The DIGE technology platform. Two different samples are derivatized with two different fluorophores, combined and then run on a single 2-D gel. Proteins are detected using a dual laser scanning device or xenon-arc-based instrument equipped with different excitation/emission filters in order to generate two separate images. The images are then matched by a computer-assisted overlay method, signals are normalized, and spots are quantified. Differences in protein expression are identified by evaluation of a pseudo-colored image and data spreadsheet. DIGE technology can maximally evaluate three different samples using Cy2-, Cy3- and Cy5-based chemistries

lE-Cy3 lE-Cy5 Excise spots; elute; digest, extract peptides; MS analyze, Protein identification

Dual Channel Imaging Technique Identification of differently expressed proteins by Dual Channel Imaging Technique (DIGE) PSHA _1_0658 IEF (pH 4-7) _1_2360 _1_0871 _1_0021 _1_0849 _2_0363 _2_0259 _2_0420 _1_2262 _1_0328 _1_1875 _1_0255 _1_2758 _1_1059 _2_0557 _1_1740 _2_0150 _1_2771 _1_0341 _1_2145 _1_3038 _2_0144 _1_0416 _1_0689 _2_0370 _1_0119 _1_3009 _1_0870 _2_0474 _1_0595 _1_3001 _1_2400 SDS PAGE - Dual Channel Imaging Technique Green label Proteome A; Red label = Proteome B

profili proteici di microrganismi patogeni come Mycobacterium avium subspecies paratuberculosis, assume rilevante importanza nello studio di patologie a carattere zoonosico Immunoproteomica dei tumori: Analisi Serologica del Proteoma (SERPA) del carcinoma

Analisi LC-MS/MS Idrolisi in situ nLC-MS/MS